AJP: Heart and Circulatory Physiology current issue

Human neutrophil peptides: a novel potential mediator of inflammatory cardiovascular diseases

Quinn, K., Henriques, M., Parker, T., Slutsky, A. S., Zhang, H. — 2008-11-06

The traditional view of atherosclerosis has recently been expanded from a predominantly lipid retentive disease to a coupling of inflammatory mechanisms and dyslipidemia. Studies have suggested a novel role for polymorphonuclear neutrophil (PMN)-dominant inflammation in the development of atherosclerosis. Human neutrophil peptides (HNPs), also known as -defensins, are secreted and released from PMN granules upon activation and are conventionally involved in microbial killing. Current evidence suggests an important immunomodulative role for these peptides. HNP levels are markedly increased in inflammatory diseases including sepsis and acute coronary syndromes. They have been found within the intima of human atherosclerotic arteries, and their deposition in the skin correlates with the severity of coronary artery diseases. HNPs form complexes with LDL in solution and increase LDL binding to the endothelial surface. HNPs have also been shown to contribute to endothelial dysfunction, lipid metabolism disorder, and the inhibition of fibrinolysis. Given the emerging relationship between PMN-dominant inflammation and atherosclerosis, HNPs may serve as a link between them and as a biological marker and potential therapeutic target in cardiovascular diseases including coronary artery diseases and acute coronary syndromes.

Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

2008-11-06

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A_2A adenosine receptors (A_2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A_2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A_2AAR^+/+ mice, A_2AAR^–/– mice, mast cell-deficient (Kit^W-sh/W-sh) mice, and chimeric mice prepared by transplanting bone marrow from A_2AAR^–/– or A_2AAR^+/+ mice to radiation-ablated A_2AAR^+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 ± 2% to 24 ± 2% and 22 ± 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A_2AAR^–/– hearts and in hearts from chimeric mice with A_2AAR^–/– bone marrow. Vehicle-treated Kit^W-sh/W-sh mice had lower IS (11 ± 3%) than WT mice, and ATL146e had no significant protective effect (16 ± 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A_2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.

Basal and IGF-I-dependent regulation of potassium channels by MAP kinases and PI3-kinase during eccentric cardiac hypertrophy

Teos, L. Y., Zhao, A., Alvin, Z., Laurence, G. G., Li, C., Haddad, G. E. — 2008-11-06

The potassium channels I_K and I_K1, responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on I_K and I_K1 through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10^–8 M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. I_K and I_K1 activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal I_K activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of I_K1 was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on I_K and I_K1. We conclude that basal I_K and I_K1 are positively maintained by steady-state Akt and ERK activities. K⁺ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K⁺ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt.

Insufficient flow reduction during LBNP in both splanchnic and lower limb areas is associated with orthostatic intolerance after bedrest

Arbeille, P., Kerbeci, P., Mattar, L., Shoemaker, J. K., Hughson, R. — 2008-11-06

We quantified the impact of a 60-day head-down tilt bed rest (HDBR) with countermeasures on the arterial response to supine lower body negative pressure (LBNP). Twenty-four women [8 control (Con), 8 exercise + LBNP (Ex-LBNP), and 8 nutrition (Nut) subjects] were studied during LBNP (0 to –45 mmHg) before (pre) and on HDBR day 55 (HDBR-55). Left ventricle diastolic volume (LVDV) and mass, flow velocities in the middle cerebral artery (MCA flow) and femoral artery (femoral flow), portal vein cross-sectional area (portal flow), and lower limb resistance (femoral resistance index) were measured. Muscle sympathetic nerve activity (MSNA) was measured in the fibular nerve. Subjects were identified as finishers or nonfinishers of the 10-min post-HDBR tilt test. At HDBR-55, LVDV, mass, and portal flow were decreased from pre-HDBR (P < 0.05) in the Con and Nut groups only. During LBNP at HDBR-55, femoral and portal flow decreased less, whereas leg MSNA increased similarly, compared with pre-HDBR in the Con, Nut, and NF groups; 11 of 13 nonfinishers showed smaller LBNP-induced reductions in both femoral and portal flow (less vasoconstriction), whereas 10 of 11 finishers maintained vasoconstriction in either one or both regions. The relative distribution of blood flow in the cerebral versus portal and femoral beds during LBNP [MCA flow/(femoral + portal flow)] increased or reduced <15% from pre-HDBR in 10 of 11 finishers but decreased >15% from pre-HDBR in 11 of 13 nonfinishers. Abnormal vasoconstriction in both the portal and femoral vascular areas was associated with orthostatic intolerance. The vascular deconditioning was partially prevented by Ex-LBNP.

Contrasting effects of presynaptic {alpha}2-adrenergic autoinhibition and pharmacologic augmentation of presynaptic inhibition on sympathetic heart rate control

2008-11-06

Presynaptic ₂-adrenergic receptors are known to exert feedback inhibition on norepinephrine release from the sympathetic nerve terminals. To elucidate the dynamic characteristics of the inhibition, we stimulated the right cardiac sympathetic nerve according to a binary white noise signal while measuring heart rate (HR) in anesthetized rabbits (n = 6). We estimated the transfer function from cardiac sympathetic nerve stimulation to HR and the corresponding step response of HR, with and without the blockade of presynaptic inhibition by yohimbine (1 mg/kg followed by 0.1 mg·kg^–1·h^–1 iv). We also examined the effect of the ₂-adrenergic receptor agonist clonidine (0.3 and 1.5 mg·kg^–1·h^–1 iv) in different rabbits (n = 5). Yohimbine increased the maximum step response (from 7.2 ± 0.8 to 12.2 ± 1.7 beats/min, means ± SE, P < 0.05) without significantly affecting the initial slope (0.93 ± 0.23 vs. 0.94 ± 0.22 beats·min^–1·s^–1). Higher dose but not lower dose clonidine significantly decreased the maximum step response (from 6.3 ± 0.8 to 6.8 ± 1.0 and 2.8 ± 0.5 beats/min, P < 0.05) and also reduced the initial slope (from 0.56 ± 0.07 to 0.51 ± 0.04 and 0.22 ± 0.06 beats·min^–1·s^–1, P < 0.05). Our findings indicate that presynaptic ₂-adrenergic autoinhibition limits the maximum response without significantly compromising the rapidity of effector response. In contrast, pharmacologic augmentation of the presynaptic inhibition not only attenuates the maximum response but also results in a sluggish effector response.

Modeling of the adrenergic response of the human IKs current (hKCNQ1/hKCNE1) stably expressed in HEK-293 cells

Imredy, J. P., Penniman, J. R., Dech, S. J., Irving, W. D., Salata, J. J. — 2008-11-06

Stable coexpression of human (h)KCNQ1 and hKCNE1 in human embryonic kidney (HEK)-293 cells reconstitutes a nativelike slowly activating delayed rectifier K⁺ current (HEK-I_Ks), allowing β-adrenergic modulation of the current by stimulation of endogenous receptors in the host cell line. HEK-I_Ks was enhanced two- to fourfold by isoproterenol (EC₅₀ = 13 nM), forskolin (10 µM), or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (50 µM), indicating an intact cAMP-dependent ion channel-regulating pathway analogous to the PKA-dependent regulation observed in native cardiac myocytes. Activation kinetics of HEK-I_Ks were accurately fit with a novel modified second-order Hodgkin-Huxley (H-H) gating model incorporating a fast and a slow gate, each independent of each other in scale and adrenergic response, or a "heterodimer" model. Macroscopically, β-adrenergic enhancement shifted the current activation threshold to more negative potentials and accelerated activation kinetics while leaving deactivation kinetics relatively unaffected. Modeling of the current response using the H-H model indicated that observed changes in gating could be explained by modulation of the opening rate of the fast gate. Under control conditions at nearly physiological temperatures (35°C), rate-dependent accumulation of HEK-I_Ks was observed only at pulse frequencies exceeding 3 Hz. Rate-dependent accumulation of I_Ks at high pulsing rate had two phases, an initial staircaselike effect followed by a slower, incremental accumulation phase. These phases are readily interpreted in the context of a heterodimeric H-H model with two independent gates with differing closing rates. In the presence of isoproterenol after normalizing for its tonic effects, rate-dependent accumulation of HEK-I_Ks appeared at lower pulse frequencies and was slightly enhanced (~25%) over control.

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice

2008-11-06

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial O₂^•– and H₂O₂ production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial O₂^•– and H₂O₂ production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular O₂^•– and H₂O₂ production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress.

Characteristics of intracellular Ca2+ cycling in intact rat heart: a comparison of sex differences

2008-11-06

Males and females show distinct differences in action potential waveform, ion channel expression patterns, and ECG characteristics. However, it is not known how sex-based differences in Ca²⁺ cycling might contribute to these differences in electrophysiological activity. The goal of this study was to investigate the differences in cellular Ca²⁺ transients in males and females and to examine how these might contribute to electrophysiological function. Ca²⁺ transients were measured in individual myocytes within microscopic regions of the fluo-4 AM-loaded left ventricular epicardium of intact rat heart of both sexes (3 to 5 mo old). Pacing protocols were used to measure transient characteristics at a basic cycle length of 500 ms and during 10-s trains of rapid pacing delivered to the left ventricular apex. Ca²⁺ transients were smaller in magnitude and longer in duration in females than in males. More importantly, the variability in Ca²⁺ transient characteristics between myocytes in a microscopic recording site (heterogeneity index) was greater for females than males for characteristics related to transient duration. The rate sensitivity of Ca²⁺ alternans development in individual myocytes was greater in females than in males, but there was also a greater heterogeneity in cellular responses to the rate dependence of alternans development in females. The longer Ca²⁺ transients in females were also associated with slower restitution, which was likely to be responsible for the development of Ca²⁺ and repolarization alternans at slower heart rates. These results demonstrate that there are distinct differences in cellular Ca²⁺ cycling in male and female rat hearts. Not only is there slower reuptake of Ca²⁺ in female rats, but there is greater local variability in Ca²⁺ cycling at the microscopic level. These sex-based differences in Ca²⁺ cycling could contribute to differences in ECG morphology and in arrhythmia sensitivity in males and females.

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

2008-11-06

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43^+/–; 66% mean reduction in Cx43) mice for 6 h at 10–15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43^+/– myocytes had significantly shorter action potential durations (APD) and increased steady-state (I_ss) and inward rectifier (I_K1) potassium currents compared with those of wild-type littermate cells. In Cx43^+/– hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of I_ss compared with unpaced Cx43^+/– hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.

Effects of clenbuterol on contractility and Ca2+ homeostasis of isolated rat ventricular myocytes

2008-11-06

Clenbuterol, a compound classified as a β₂-adrenoceptor (AR) agonist, has been employed in combination with left ventricular assist devices (LVADs) to treat patients with severe heart failure. Previous studies have shown that chronic administration of clenbuterol affects cardiac excitation-contraction coupling. However, the acute effects of clenbuterol and the signaling pathway involved remain undefined. We investigated the acute effects of clenbuterol on isolated ventricular myocyte sarcomere shortening, Ca²⁺ transients, and L-type Ca²⁺ current and compared these effects to two other clinically used β₂-AR agonists: fenoterol and salbutamol. Clenbuterol (30 µM) produced a negative inotropic response, whereas fenoterol showed a positive inotropic response. Salbutamol had no significant effects. Clenbuterol reduced Ca²⁺ transient amplitude and L-type Ca²⁺ current. Selective β₁-AR blockade did not affect the action of clenbuterol on sarcomere shortening but significantly reduced contractility in the presence of fenoterol and salbutamol (P < 0.05). Incubation with 2 µg/ml pertussis toxin significantly reduced the negative inotropic effects of 30 µM clenbuterol. In addition, overexpression of inhibitory G protein (G_i) by adenoviral transfection induced a stronger clenbuterol-mediated negative inotropic effect, suggesting the involvement of the G_i protein. We conclude that clenbuterol does not increase and, at high concentrations, significantly depresses contractility of isolated ventricular myocytes, an effect not seen with fenoterol or salbutamol. In its negative inotropism, clenbuterol predominantly acts through G_i, and the consequent downstream signaling pathways activation may explain the beneficial effects observed during chronic administration of clenbuterol in patients treated with LVADs.

Heterogeneity in conduit artery function in humans: impact of arterial size

2008-11-06

To determine whether conduit artery size affects functional responses, we compared the magnitude, time course, and eliciting shear rate stimulus for flow-mediated dilation (FMD) in healthy men (n = 20; 31 ± 7 yr). Upper limb (brachial and radial) and lower limb (common and superficial femoral) FMD responses were simultaneously assessed, whereas popliteal responses were measured in the same subjects during a separate visit. Glyceryl trinitrate (GTN)-mediated responses were similarly examined. Edge detection and wall tracking of high-resolution B-mode arterial ultrasound images, combined with synchronized Doppler waveform envelope analysis, were used to calculate conduit artery diameter, blood flow, and shear rate continuously across the cardiac cycle. Baseline artery size correlated inversely with the FMD response (r = –0.57, P < 0.001). Within-artery comparisons revealed a significant inverse correlation between artery size and FMD% for the radial (r = –0.66, P = 0.001), brachial (r = –0.55, P = 0.01), and popliteal artery (r = –0.48, P = 0.03), but not for the superficial and common femoral artery. Normalization of FMD responses for differences in eliciting shear rate did not abolish the between-artery relationship for artery function and size (r = –0.48, P < 0.001), suggesting that differences between artery function responses were not entirely due to size-related differences in shear rate. This was reinforced by a significant between-artery correlation for GTN responses and baseline artery size (r = –0.74, P < 0.001). In summary, systematic differences exist in vascular function responses of conduit arteries that differ in size. This raises the possibility that differences in artery size within or between individuals may influence functional responses.

Inhibition of rat aortic smooth muscle contraction by 2-methoxyestradiol

Gui, Y., Zheng, X.-L., Zheng, J., Walsh, M. P. — 2008-11-06

Recent studies suggest that 2-methoxyestradiol (2-ME), an estrogen metabolite, has a similar inhibitory effect as 17β-estradiol (E₂) on vascular tone. However, it is not known whether 2-ME mediates the effects of E₂ or by what mechanism 2-ME regulates smooth muscle contraction. Therefore, we compared the effects of 2-ME and E₂ on rat aortic smooth muscle contraction. A preincubation with 2-ME (10 µM) for 1 h inhibited phenylephrine (PE)-induced tension in endothelium-intact, but not -denuded, tissues, whereas E₂ inhibited PE-induced contraction in both preparations. The effects of 2-ME and E₂ on endothelium-intact preparations were prevented by l-NAME hydrochloride (a nitric oxide synthase inhibitor). The 2-ME treatment reduced PE-induced phosphorylation of the 20-kDa myosin regulatory light chain. The inhibitory effects of 2-ME and E₂ were not affected by ICI-182780 (an estrogen receptor antagonist) or actinomycin D (a gene transcription inhibitor); however, the effect of 2-ME, but not E₂, was prevented by cycloheximide (a protein synthesis inhibitor). Furthermore, the effect of E₂ was not blocked by 1-aminobenzotriazole (a cytochrome P-450 inhibitor) or Ro 41-0960 (a catechol-O-methyltransferase inhibitor). The effect of 2-ME was not mimicked by microtubule-interfering agents (nocodazole or Taxol). We conclude that 2-ME inhibits smooth muscle contractility through an endothelium- and nitric oxide-dependent mechanism, which does not involve estrogen receptors or microtubule disruption. The effect of 2-ME, but not E₂, involves de novo protein synthesis. 2-ME does not mediate the inhibitory effect of E₂ on smooth muscle contraction. These results support a potentially important role of 2-ME in the regulation of smooth muscle tone in the vasculature.

Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole

Mukhopadhyay, S., Lee, J., Sehgal, P. B. — 2008-11-06

Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by ~50% in Golgi membranes after MCTP despite increases in -SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70–95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.

ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes

Liu, J., Mao, W., Ding, B., Liang, C.-s. — 2008-11-06

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis- expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT- blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.

Laminar shear stress inhibits lipid peroxidation induced by high glucose plus arachidonic acid in endothelial cells

Mun, G. I., An, S. M., Park, H., Jo, H., Boo, Y. C. — 2008-11-06

Elevated blood glucose and free fatty acids induce oxidative stress associated with the incidence of cardiovascular disease. In contrast, laminar shear stress (LSS) plays a critical role in maintaining vascular health. The present study examined the mechanism for the antioxidant effect of LSS attenuating the oxidative stress induced by high glucose (HG) and arachidonic acid (AA) in human umbilical vein endothelial cells. HG and AA synergistically decreased cell viability and increased glutathione (GSH) oxidation and lipid peroxidation. The lipid peroxidation was markedly prevented by LSS as well as tetrahydrobiopterin (BH₄) and GSH. LSS increased BH₄ and GSH contents, and expression of GTP cyclohydrolase-1 and glutamylcysteine ligase (GCL) involved in their biosynthesis. Inhibition of GCL activity by DL-buthionine-(S,R)-sulfoximine and small-interfering RNA-mediated knockdown of GCL lessened the antioxidant effect of LSS. Therefore, it is suggested that LSS enhances antioxidant capacity of endothelial cells and thereby attenuates the oxidative stress caused by cardiovascular risk factors.

Chronic central leptin infusion restores cardiac sympathetic-vagal balance and baroreflex sensitivity in diabetic rats

do Carmo, J. M., Hall, J. E., da Silva, A. A. — 2008-11-06

This study tested whether leptin restores sympathetic-vagal balance, heart rate (HR) variability, and cardiac baroreflex sensitivity (BRS) in streptozotocin (STZ)-induced diabetes. Sprague-Dawley rats were instrumented with arterial and venous catheters, and a cannula was placed in the lateral ventricle for intracerebroventricular (ICV) leptin infusion. Blood pressure (BP) and HR were monitored by telemetry. BRS and HR variability were estimated by linear regression between HR and BP responses to phenylephrine or sodium nitroprusside and autoregressive spectral analysis. Measurements were made during control period, 7 days after induction of diabetes, and 7 days after ICV leptin infusion. STZ diabetes was associated with hyperglycemia (422 ± 17 mg/dl) and bradycardia (–79 ± 4 beats/min). Leptin decreased glucose levels (165 ± 16 mg/dl) and raised HR to control values (303 ± 10 to 389 ± 10 beats/min). Intrinsic HR (IHR) and chronotropic responses to a full-blocking dose of propranolol and atropine were reduced during diabetes (260 ± 7 vs. 316 ± 6, –19 ± 2 vs. –43 ± 6, and 39 ± 3 vs. 68 ± 8 beats/min), and leptin treatment restored these variables to normal (300 ± 7, –68 ± 10, and 71 ± 8 beats/min). Leptin normalized BRS (bradycardia, –2.6 ± 0.3, –1.7 ± 0.2, and –3.0 ± 0.5; and tachycardia, –3.2 ± 0.4, –1.9 ± 0.3, and –3.4 ± 0.3 beats·min^–1·mmHg^–1 for control, diabetes, and leptin) and HR variability (23 ± 4 to 11 ± 1.5 ms²). Chronic glucose infusion to maintain hyperglycemia during leptin infusion did not alter the effect of leptin on IHR but abolished the improved BRS. These results show rapid impairment of autonomic nervous system control of HR after the induction of diabetes and that central nervous system actions of leptin can abolish the hyperglycemia as well as the altered IHR and BRS in STZ-induced diabetes.

Role of EDHF in type 2 diabetes-induced endothelial dysfunction

Park, Y., Capobianco, S., Gao, X., Falck, J. R., Dellsperger, K. C., Zhang, C. — 2008-11-06

Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in modulating vasomotor tone, especially in microvessels when nitric oxide-dependent control is compromised such as in diabetes. Epoxyeicosatrienoic acids (EETs), potassium ions (K⁺), and hydrogen peroxide (H₂O₂) are proposed as EDHFs. However, the identity (or identities) of EDHF-dependent endothelial dilators has not been clearly elucidated in diabetes. We assessed the mechanisms of EDHF-induced vasodilation in wild-type (WT, normal), db/db (advanced type 2 diabetic) mice, and db/db mice null for TNF (db^TNF–/db^TNF–). In db/db mice, EDHF-induced vasodilation [ACh-induced vasodilation in the presence of N^G-nitro-l-arginine methyl ester (l-NAME, 10 µmol/l) and prostaglandin synthase inhibitor indomethacin (Indo, 10 µmol/l)] was diminished after the administration of catalase (an enzyme that selectively dismutates H₂O₂ to water and oxygen, 1,000 U/ml); administration of the combination of charybdotoxin (a nonselective blocker of intermediate-conductance Ca²⁺-activated K⁺ channels, 10 µmol/l) and apamin (a selective blocker of small-conductance Ca²⁺-activated K⁺ channels, 50 µmol/l) also attenuated EDHF-induced vasodilation, but the inhibition of EETs synthesis [14,15-epoxyeicosa-5(Z)-enoic acid; 10 µmol/l] did not alter EDHF-induced vasodilation. In WT controls, EDHF-dependent vasodilation was significantly diminished after an inhibition of K⁺ channel, EETs synthesis, or H₂O₂ production. Our molecular results indicate that mRNA and protein expression of interleukin-6 (IL-6) were greater in db/db versus WT and db^TNF–/db^TNF– mice, but neutralizing antibody to IL-6 (anti-IL-6; 0.28 mg·ml^–1·kg^–1 ip for 3 days) attenuated IL-6 expression in db/db mice. The incubation of the microvessels with IL-6 (5 ng/ml) induced endothelial dysfunction in the presence of l-NAME and Indo in WT mice, but anti-IL-6 restored ACh-induced vasodilation in the presence of l-NAME and Indo in db/db mice. In db^TNF–/db^TNF– mice, EDHF-induced vasodilation was greater and comparable with controls, but IL-6 decreased EDHF-mediated vasodilation. Our results indicate that EDHF compensates for diminished NO-dependent dilation in IL-6-induced endothelial dysfunction by the activation of H₂O₂ or a K⁺ channel in type 2 diabetes.

Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery: pharmacology and implication for spontaneous contractility

von der Weid, P.-Y., Rahman, M., Imtiaz, M. S., van Helden, D. F. — 2008-11-06

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca²⁺ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca²⁺ released from Ca²⁺ stores, we observed that intracellular Ca²⁺ transients accompanied some STDs, although there were many exceptions where Ca²⁺ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP₃Rs) but not by treatments known to alter Ca²⁺ release via ryanodine receptors. A role for Ca²⁺-activated Cl^– (Cl_Ca) channels was indicated, as STDs were dependent on the Cl^– but not Na⁺ concentration of the superfusing solution and were inhibited by the Cl_Ca channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl^– blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd³⁺, and Ni²⁺ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd³⁺, and Ni²⁺ altered the occurrence of spontaneous APs. Our findings support a role for Ca²⁺ release through IP₃Rs and a resultant opening of Cl_Ca channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.

Dissection of two Cx37-independent conducted vasodilator mechanisms by deletion of Cx40: electrotonic versus regenerative conduction

Figueroa, X. F., Duling, B. R. — 2008-11-06

Conduction of changes in diameter plays an important role in the coordination of peripheral vascular resistance and, thereby, in the control of arterial blood pressure. It is thought that conduction of vasomotor signals relies on the electrotonic spread of changes in membrane potential from a site of stimulation through gap junctions connecting the cells of the vessel wall. To explore this idea, we stimulated a short segment of mouse cremasteric arterioles with an application, via micropipette, of ACh, an endothelium-dependent vasodilator, or pinacidil, an ATP-sensitive K⁺ channel opener. Vasodilations were evaluated at the stimulation site (local) and at 500, 1,000, and 2,000 µm upstream. The vasodilator response evoked by direct arteriolar hyperpolarization induced by pinacidil decayed rapidly with distance, as expected for the passive spread of an electrical signal. Deletion of the gap junction proteins connexin37 or connexin40 did not alter the conduction of pinacidil-induced vasodilation. In contrast to pinacidil, the vasodilator response activated by ACh spread along the entire vessel without decrement. Although the ACh-induced conducted vasodilation was similar in wild-type and connexin37 knockout mice, deletion of connexin40 converted the nondecremental conducted response activated by ACh into one similar to that of pinacidil, with a decline in magnitude along the vessel length. These results suggest that ACh activates a mechanism of regenerative conduction of vasodilator responses. Connexin40 is essential for the ACh-activated regenerative vasodilator mechanism. However, neither connexin40 nor connexin37 is indispensable for the electrotonic spread of hyperpolarizing signals.

Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Zima, A. V., Qin, J., Fill, M., Blatter, L. A. — 2008-11-06

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 µM) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 µM) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1–1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability (P_o) in a dose-dependent fashion (dissociation constant = 4.2 µM). High concentrations of AMT (>25 µM) evoked frequent long openings with P_o reaching very high levels (>0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity.

Impaired Ca2+ homeostasis is associated with atrial fibrillation in the {alpha}1D L-type Ca2+ channel KO mouse

Mancarella, S., Yue, Y., Karnabi, E., Qu, Y., El-Sherif, N., Boutjdir, M. — 2008-11-06

The novel _1D Ca²⁺ channel together with _1C Ca²⁺ channel contribute to the L-type Ca²⁺ current (I_Ca-L) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the _1D gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the _1D Ca²⁺ channel to the global I_Ca-L, the intracellular Ca²⁺ transient, the Ca²⁺ handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from _1D KO mice demonstrated significant sinus bradycardia, atrioventricular block, and vulnerability to AF. The wild-type mice showed no ECG abnormalities and no AF. Patch-clamp recordings from isolated _1D KO atrial myocytes revealed a significant reduction of I_Ca-L (24.5%; P < 0.05). However, there were no changes in other currents such as I_Na, I_Ca-T, I_K, I_f, and I_to and no changes in _1C mRNA levels of _1D KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular Ca²⁺ transient (~40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR Ca²⁺ content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR Ca²⁺ release in _1D KO atria. In conclusion, genetic deletion of _1D Ca²⁺ channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the _1D KO mice were associated with a decrease in the total I_Ca-L density, a reduction in intracellular Ca²⁺ transient, and impaired intracellular Ca²⁺ handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the _1D KO mice.

Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion

Kubli, D. A., Quinsay, M. N., Huang, C., Lee, Y., Gustafsson, A. B. — 2008-11-06

Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) is a member of the Bcl-2 homology domain 3-only subfamily of proapoptotic Bcl-2 proteins and is associated with cell death in the myocardium. In this study, we investigated the potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT-sensitive homodimer that increased after myocardial ischemia-reperfusion (I/R). The presence of the antioxidant N-acetylcysteine reduced I/R-induced homodimerization of Bnip3. Overexpression of Bnip3 in cells revealed that most of exogenous Bnip3 exists as a DTT-sensitive homodimer that correlated with increased cell death. In contrast, endogenous Bnip3 existed mainly as a monomer under normal conditions in the heart. Screening of the Bnip3 protein sequence revealed a single conserved cysteine residue at position 64. Mutation of this cysteine to alanine (Bnip3C64A) or deletion of the NH₂-terminus (amino acids 1-64) resulted in reduced cell death activity of Bnip3. Moreover, mutation of a histidine residue in the COOH-terminal transmembrane domain to alanine (Bnip3H173A) almost completely inhibited the cell death activity of Bnip3. Bnip3C64A had a reduced ability to interact with Bnip3, whereas Bnip3H173A was completely unable to interact with Bnip3, suggesting that homodimerization is important for Bnip3 function. A consequence of I/R is the production of reactive oxygen species and oxidation of proteins, which promotes the formation of disulfide bonds between proteins. Thus, these experiments suggest that Bnip3 functions as a redox sensor where increased oxidative stress induces homodimerization and activation of Bnip3 via cooperation of the NH₂-terminal cysteine residue and the COOH-terminal transmembrane domain.

Comparison of baroreceptive to other afferent synaptic transmission to the medial solitary tract nucleus

Andresen, M. C., Peters, J. H. — 2008-11-06

Cranial nerve visceral afferents enter the brain stem to synapse on neurons within the solitary tract nucleus (NTS). The broad heterogeneity of both visceral afferents and NTS neurons makes understanding afferent synaptic transmission particularly challenging. To study a specific subgroup of second-order neurons in medial NTS, we anterogradely labeled arterial baroreceptor afferents of the aortic depressor nerve (ADN) with lipophilic fluorescent tracer (i.e., ADN+) and measured synaptic responses to solitary tract (ST) activation recorded from dye-identified neurons in medial NTS in horizontal brain stem slices. Every ADN+ NTS neuron received constant-latency ST-evoked excitatory postsynaptic currents (EPSCs) (jitter <192 µs, SD of latency). Stimulus-recruitment profiles showed single thresholds and no suprathreshold recruitment, findings consistent with EPSCs arising from a single, branched afferent axon. Frequency-dependent depression of ADN+ EPSCs averaged ~70% for five shocks at 50 Hz, but single-shock failure rates did not exceed 4%. Whether adjacent ADN– or those from unlabeled animals, other second-order NTS neurons (jitters <200 µs) had ST transmission properties indistinguishable from ADN+. Capsaicin (CAP; 100 nM) blocked ST transmission in some neurons. CAP-sensitive ST-EPSCs were smaller and failed over five times more frequently than CAP-resistant responses, whether ADN+ or from unlabeled animals. Variance-mean analysis of ST-EPSCs suggested uniformly high probabilities for quantal glutamate release across second-order neurons. While amplitude differences may reflect different numbers of contacts, higher frequency-dependent failure rates in CAP-sensitive ST-EPSCs may arise from subtype-specific differences in afferent axon properties. Thus afferent transmission within medial NTS differed by axon class (e.g., CAP sensitive) but was indistinguishable by source of axon (e.g., baroreceptor vs. nonbaroreceptor).

PPADS does not block contraction-induced prostaglandin E2 synthesis in cat skeletal muscle

McCord, J. L., Hayes, S. G., Kaufman, M. P. — 2008-11-06

Pyridoxal-phosphate-6-azophenyl-2'-4-disulfonate (PPADS), a purinergic 2 (P₂) receptor antagonist, has been shown to attenuate the exercise pressor reflex in cats. In vitro, however, PPADS has been shown to block the production of prostaglandins, some of which play a role in evoking the exercise pressor reflex. Thus the possibility exists that PPADS blocks the exercise pressor reflex through a reduction in prostaglandin synthesis rather than through the blockade of P₂ receptors. Using microdialysis, we collected interstitial fluid from skeletal muscle to determine prostaglandin E₂ (PGE₂) concentrations during the intermittent contraction of the triceps surae muscle before and after a popliteal arterial injection of PPADS (10 mg/kg). We found that the PGE₂ concentration increased in response to the intermittent contraction before and after the injection of PPADS (both, P < 0.05). PPADS reduced the pressor response to exercise (P < 0.05) but had no effect on the magnitude of PGE₂ production during contraction (P = 0.48). These experiments demonstrate that PPADS does not block the exercise pressor reflex through a reduction in PGE₂ synthesis. We suggest that PGE₂ and P₂ receptors play independent roles in stimulating the exercise pressor reflex.

Effects of rosuvastatin on cardiovascular morphology and function in an ApoE-knockout mouse model of atherosclerosis

2008-11-06

This study investigated the effects of rosuvastatin on plaque progression and in vivo coronary artery function in apolipoprotein E-knockout (ApoE-KO) mice, using noninvasive high-resolution ultrasound techniques. Eight-week-old male ApoE-KO mice (n = 20) were fed a high-fat diet with or without rosuvastatin (10 µmol·kg^–1·day^–1) for 16 wk. When compared with control, rosuvastatin reduced total cholesterol levels (P < 0.05) and caused significant retardation of lesion progression in the brachiocephalic artery, as visualized in vivo using an ultrasound biomicroscope (P < 0.05). Histological analysis confirmed the reduction of brachiocephalic atherosclerosis and also revealed an increase in collagen content in the statin-treated group (P < 0.05). Coronary volumetric flow was measured by simultaneous recording of Doppler velocity signals and left coronary artery morphology before and during adenosine infusion. The hyperemic flow in response to adenosine was significantly greater in left coronary artery following 16 wk of rosuvastatin treatment (P < 0.001), whereas the baseline flow was similar in both groups. In conclusion, rosuvastatin reduced brachiocephalic artery atherosclerotic plaques in ApoE-KO mice. Coronary artery function assessed using recently developed in vivo ultrasound-based protocols, also improved.

Increased diastolic time fraction as beneficial adjunct of {alpha}1-adrenergic receptor blockade after percutaneous coronary intervention

Kolyva, C., Verhoeff, B.-J., Spaan, J. A. E., Piek, J. J., Siebes, M. — 2008-11-06

The effect of ₁-receptor blockade with urapidil on coronary blood flow and left ventricular function has been attributed to relief of diffuse coronary vasoconstriction following percutaneous coronary intervention (PCI). We hypothesized that an increase in diastolic time fraction (DTF) contributes to the beneficial action of urapidil. In eleven patients with a 63% (SD 13) diameter stenosis, ECG, aortic pressure (P_a) and distal intracoronary pressure (P_d), and blood flow velocity were recorded at baseline and throughout adenosine-induced hyperemia. Measurements were obtained before and after PCI and after subsequent ₁-receptor blockade with urapidil (10 mg ic). DTF was determined from the ECG and the P_a waveform. Functional parameters such as coronary flow velocity reserve, fractional flow reserve, and an index of hyperemic microvascular resistance (HMR) were assessed. Urapidil administration after PCI induced an upward shift in the DTF-heart rate relationship, resulting in a 3.1% (SD 2.7) increase in hyperemic DTF at a constant heart rate (P < 0.005) due to a shorter duration of systole. Hyperemic P_a and P_d decreased, respectively, by 6.1% (SD 6.6; P < 0.05) and 5.7% (SD 5.8; P < 0.01) after ₁-blockade. Although epicardially measured functional parameters were on average not altered by ₁-blockade due to concurrent changes in pressure and heart rate, HMR decreased by urapidil in those patients where coronary pressure remained constant. In conclusion, ₁-receptor blockade after PCI produced a modest but significant prolongation of DTF at a given heart rate, thereby providing an adjunctive beneficial mechanism for improving subendocardial perfusion, which critically depends on DTF.

Role of estrogen receptor subtypes in estrogen-induced organ-specific vasorelaxation after trauma-hemorrhage

Ba, Z. F., Chaudry, I. H. — 2008-11-06

Although endothelin-1 (ET-1)-induced organ hypoperfusion after trauma-hemorrhage is improved by estrogen administration, it remains unclear whether estrogen receptor (ER) subtypes play any role in the attenuation of ET-1-induced vasoconstriction in any specific organ bed. To investigate this, isolated perfusion experiments in the heart, liver, small intestine, kidney, and lung were carried out in sham, at the time of maximum bleedout (MBO; i.e., 5-cm midline incision, with removal of 60% of circulating blood volume over 45 min to maintain a mean blood pressure of 40 mmHg), and 2 h after trauma-hemorrhage and resuscitation (T-H/R). Organ-specific ET-1-induced vasoconstriction was evaluated, and the effects of 17β-estradiol (E₂) and ER-specific agonists propylpyrazole triol (PPT; ER agonist) and diarylpropionitrile (DPN; ERβ agonist) were determined. ET-1 induced the greatest vasoconstriction in sham animals, with the strongest response in the kidneys, followed by the small intestine and liver. ET-1-induced responses were weakest in the heart and lungs. ET-1-induced vasoconstriction was evident at the time of MBO but was significantly decreased at 2 h after T-H/R. ERβ plays an important role in cardiac performance, as evidenced by improved heart performance (+dP/dt) in the presence of DPN. DPN also induced a greater effect than PPT in the reduction of ET-1-induced vasoconstriction in the kidneys and lungs. In contrast, PPT attenuated ET-1-induced vasoconstriction in the liver, whereas both DPN and PPT were equally effective in the small intestine. The increased +dP/dt values induced by E₂, DPN, or PPT were evident at the time of MBO but were significantly decreased at 2 h after T-H/R. These data indicate that the effects of ET-1 on vasoconstriction and the role of ER subtypes in estrogen-induced vasorelaxation are organ specific and temporally specific after trauma-hemorrhage.

Role of CYP epoxygenases in A2AAR-mediated relaxation using A2AAR-null and wild-type mice

2008-11-06

We hypothesized that A_2A adenosine receptor (A_2AAR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A_2AAR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5'-N-ethylcarboxamide (NECA; 10^–6 M), an adenosine analog, caused relaxation in wild-type A_2AAR (A_2AAR^+/+; +33.99 ± 4.70%, P < 0.05) versus contraction in A_2AAR knockout (A_2AAR^–/–; –27.52 ± 4.11%) mouse aortae. An A_2AAR-specific antagonist (SCH-58261; 1µM) changed the NECA (10^–6 M) relaxation response to contraction (–35.82 ± 4.69%, P < 0.05) in A_2AAR^+/+ aortae, whereas no effect was noted in A_2AAR^–/– aortae. Significant contraction was seen in the absence of the endothelium in A_2AAR^+/+ (–2.58 ± 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitro-l-arginine methyl ester; 100 µM) and a cyclooxygenase inhibitor (indomethacin; 10 µM) failed to block NECA-induced relaxation in A_2AAR^+/+ aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 µM) changed NECA-mediated relaxation (–22.74 ± 5.11% at 10^–6 M) and CGS-21680-mediated relaxation (–18.54 ± 6.06% at 10^–6 M) to contraction in A_2AAR^+/+ aortae, whereas no response was noted in A_2AAR^–/– aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 µM] was able to block NECA-induced relaxation in A_2AAR^+/+ aortae, whereas -hydroxylase inhibitors (10 µM dibromo-dodecenyl-methylsulfimide and 10 µM HET-0016) changed contraction into relaxation in A_2AAR^–/– aorta. Cyp2c29 protein was upregulated in A_2AAR^+/+ aortae, whereas Cyp4a was upregulated in A_2AAR^–/– aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A_2AAR^+/+ versus A_2AAR^–/– aortae. EET levels were not significantly different between A_2AAR^+/+ and A_2AAR^–/– aortae. It is concluded that CYP epoxygenases play an important role in A_2AAR-mediated relaxation, and the deletion of the A_2AAR leads to contraction through Cyp4a.

Ser9 phosphorylation of mitochondrial GSK-3{beta} is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

2008-11-06

The aim of this study was to determine the role of GSK-3β in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3β and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H₂O₂ from 38.3 ± 2.7% to 26.0 ± 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3β knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3β (K85R). The level of colocalization of intracellular GSK-3β with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3β or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3β colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H₂O₂ was inhibited by EPO pretreatment and by GSK-3β knockdown. These results suggest that the suppression of GSK-3β activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.

Fluid shear stress modulates endothelial cell invasion into three-dimensional collagen matrices

Kang, H., Bayless, K. J., Kaunas, R. — 2008-11-06

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of ~5 dyn/cm², whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm² shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.

Reverse changes in cardiac substrate oxidation in dogs recovering from heart failure

2008-11-06

When recovering from heart failure (HF), the myocardium displays a marked plasticity and can regain normal gene expression and function; however, recovery of substrate oxidation capacity has not been explored. We tested whether cardiac functional recovery is matched by normalization of energy substrate utilization during post-HF recovery. HF was induced in dogs by pacing the left ventricle (LV) at 210–240 beats/min for 4 wk. Tachycardia was discontinued, and the heart was allowed to recover. An additional group was studied in HF, and healthy dogs served as controls (n = 8/group). Cardiac free fatty acids (FFAs) and glucose oxidation were measured with [³H]oleate and [¹⁴C]glucose. At 10 days of recovery, hemodynamic parameters returned to control values; however, the contractile response to dobutamine remained depressed, LV end-diastolic volume was 28% higher than control, and the heart mass-to-body mass ratio was increased (9.8 ± 0.4 vs. 7.5 ± 0.2 g/kg, P < 0.05). HF increased glucose oxidation (76.8 ± 19.7 nmol·min^–1·g^–1) and decreased FFA oxidation (20.7 ± 6.4 nmol·min^–1·g^–1), compared with normal dogs (24.5 ± 6.3 and 51.7 ± 9.6 nmol·min^–1·g^–1, respectively), and reversed to normal values at 10 days of recovery (25.4 ± 6.0 and 46.6 ± 6.7 nmol·min^–1·g^–1, respectively). However, similar to HF, the recovered dogs failed to increase glucose and fatty acid uptake in response to pacing stress. The activity of myocardial citrate synthase and aconitase was significantly decreased during recovery compared with that in control dogs (58 and 27% lower, respectively, P < 0.05), indicating a persistent reduction in mitochondrial oxidative capacity. In conclusion, cardiac energy substrate utilization is normalized in the early stage of post-HF recovery at baseline, but not under stress conditions.

No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning

2008-11-06

Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1, PP2A, and PP2B and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Göttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 ± 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 ± 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2A and PP1, but not PP2B, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2A but not with PP1. Although the total PP2A immunoreactivity (confocal laser scan) was increased to 154 ± 24% and 194 ± 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2A coimmunoprecipitated with Cx43 remained unchanged. Only PP2A coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2A is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.

TGF-{beta}1 is a negative regulator of lymphatic regeneration during wound repair

2008-11-06

Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -β₁ (TGF-β₁) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-β₁ on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-β₁ expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-β₁ on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-β₁, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-β₁ to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-β₁ inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-β₁ caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-β₁ during wound repair resulted in lymphatic fibrosis and the coexpression of -smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-β₁ expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-β₁ expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-β₁ expression may be useful in promoting more rapid lymphatic regeneration.

Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

2008-11-06

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.

High glucose increases the expression of Gq/11{alpha} and PLC-{beta} proteins and associated signaling in vascular smooth muscle cells

Descorbeth, M., Anand-Srivastava, M. B. — 2008-11-06

The levels and activity of protein kinase C and diacylglycerol were shown to be upregulated in diabetes/hyperglycemia; however, studies on the expression of upstream signaling molecules of phosphatidylinositol turnover were lacking. The present study was therefore undertaken to examine whether hyperglycemia/diabetes could also modulate the expression of G_q and phospholipase C-β (PLC-β) proteins and associated phosphatidylinositol turnover signaling in aortic vascular smooth muscle cells (VSMCs) and A10 VSMCs exposed to high glucose. Aortic VSMCs from streptozotocin-diabetic rats exhibited an increased expression of G_q and PLC-β₁ proteins (60% and 30%, respectively) compared with control cells as determined by Western blot analysis. The pretreatment of A10 VSMCs with high glucose (26 mM) for 3 days also augmented the levels of G_q, G11, PLC-β₁ and -β₂ proteins by about 50, 35, 30, and 30%, respectively, compared with control cells that were restored to control levels by endothelin-1 (ET-1), ET types A and B (ET_A and ET_B) receptors, and angiotensin II type 1 (AT₁) receptor antagonists. In addition, ET-1-stimulated inositol triphosphate formation was also significantly higher in VSMCs exposed to high glucose, whereas the basal levels of inositol triphosphate were not different between the two groups. Furthermore, the treatment of A10 VSMCs with angiotensin II and ET-1 also significantly increased the levels of G_q/11 and PLC-β proteins that were restored toward control levels by ET_A/ET_B and AT₁ receptor antagonists. These results suggest that high glucose augments the expression of G_q/11, PLC-β, and mediated signaling in VSMCs, which may be attributed to AT₁, ET_A, and ET_B receptors.

Nitrite consumption in ischemic rat heart catalyzed by distinct blood-borne and tissue factors

McNulty, P. H., Scott, S., Kehoe, V., Kozak, M., Sinoway, L. I., Li, J. — 2008-11-06

Nitric oxide (NO) may limit myocardial ischemia-reperfusion injury by slowing the mitochondrial metabolism. We examined whether rat heart contains catalysts potentially capable of reducing nitrite to NO during an episode of regional myocardial ischemia produced by temporary coronary artery occlusion. In intact Sprague-Dawley rats, a 15-min coronary occlusion lowered the nitrite concentration of the myocardial regions exhibiting ischemic glucose metabolism to ~50% that of nonischemic regions (185 ± 223 vs. 420 ± 203 nmol/l). Nitrite was rapidly repleted during subsequent reperfusion. The heart tissue tested in vitro acquired a substantial ability to consume nitrite when made hypoxic at neutral pH, and this ability was slightly enhanced by simultaneously lowering the pH to 5.5. More than 70% of this activity could be abolished by flushing the coronary circulation with crystalloid to remove trapped erythrocytes. Correspondingly, erythrocytes demonstrated the ability to reduce exogenous nitrite to NO under hypoxic conditions in vitro. In erythrocyte-free heart tissue, the nitrite consumption increased fivefold when the pH was lowered to 5.5. Approximately 40% of this pH-sensitive increase in nitrite consumption could be blocked by the xanthine oxidoreductase inhibitor allopurinol, whereas lowering the Po₂ sufficiently to desaturate myoglobin accelerated it further. We conclude that rat heart contains several factors capable of catalyzing ischemic nitrite reduction; the most potent is contained within erythrocytes and activated by hypoxia, whereas the remainder includes xanthine oxidoreductase and other pH-sensitive factors endogenous to heart tissue, including deoxymyoglobin.

Ranolazine combined with enalapril or metoprolol prevents progressive LV dysfunction and remodeling in dogs with moderate heart failure

2008-11-06

Acute intravenous infusion of ranolazine (Ran), an anti-ischemic/antiangina drug, was previously shown to improve left ventricular (LV) ejection fraction (EF) without a concomitant increase in myocardial oxygen consumption in dogs with chronic heart failure (HF). This study examined the effects of treatment with Ran alone and in combination with metoprolol (Met) or enalapril (Ena) on LV function and remodeling in dogs with HF. Dogs (n = 28) with microembolization-induced HF were randomized to 3 mo oral treatment with Ran alone [375 mg twice daily (bid); n = 7], Ran (375 mg bid) in combination with Met tartrate (25 mg bid; n = 7), Ran (375 mg bid) in combination with Ena (10 mg bid; n = 7), or placebo (PL; Ran vehicle bid; n = 7). Ventriculographic measurements of LV end-diastolic volume (EDV) and end-systolic volume (ESV) and LV EF were obtained before treatment and after 3 mo of treatment. In PL-treated dogs, EDV and ESV increased significantly. Ran alone prevented the increase in EDV and ESV seen in the PL group and significantly increased EF, albeit modestly, from 35 ± 1% to 37 ± 2%. When combined with either Ena or Met, Ran prevented the increase in EDV, significantly decreased ESV, and markedly increased EF compared with those of PL. EF increased from 35 ± 1% to 40 ± 1% with Ran + Ena and from 34 ± 1% to 41 ± 1% with Ran + Met. Ran alone or in combination with Ena or Met was also associated with beneficial effects at the cellular level on histomorphometric parameters such as hypertrophy, fibrosis, and capillary density as well as the expression for pathological hypertrophy and Ca²⁺ cycling genes. In conclusion, Ran prevented progressive LV dysfunction and global and cellular myocardial remodeling, and Ran in combination with Ena or Met improved LV function beyond that observed with Ran alone.

PER3 polymorphism and cardiac autonomic control: effects of sleep debt and circadian phase

Viola, A. U., James, L. M., Archer, S. N., Dijk, D.-J. — 2008-11-06

A variable number tandem repeat polymorphism in the coding region of the circadian clock PERIOD3 (PER3) gene has been shown to affect sleep. Because circadian rhythms and sleep are known to modulate sympathovagal balance, we investigated whether homozygosity for this PER3 polymorphism is associated with changes in autonomic nervous system (ANS) activity during sleep and wakefulness at baseline and after sleep deprivation. Twenty-two healthy participants were selected according to their PER3 genotype. ANS activity, evaluated by heart rate (HR) and HR variability (HRV) indexes, was quantified during baseline sleep, a 40-h period of wakefulness, and recovery sleep. Sleep deprivation induced an increase in slow-wave sleep (SWS), a decrease in the global variability, and an unbalance of the ANS with a loss of parasympathetic predominance and an increase in sympathetic activity. Individuals homozygous for the longer allele (PER3^5/5) had more SWS, an elevated sympathetic predominance, and a reduction of parasympathetic activity compared with PER3^4/4, in particular during baseline sleep. The effects of genotype were strongest during non-rapid eye movement (NREM) sleep and absent or much smaller during REM sleep. The NREM-REM cycle-dependent modulation of the low frequency-to-(low frequency + high frequency) ratio was diminished in PER3^5/5 individuals. Circadian phase modulated HR and HRV, but no interaction with genotype was observed. In conclusion, the PER3 polymorphism affects the sympathovagal balance in cardiac control in NREM sleep similar to the effect of sleep deprivation.

Ischemia-reperfusion injury in rats affects hydraulic conductivity in two phases that are temporally and mechanistically separate

Victorino, G. P., Ramirez, R. M., Chong, T. J., Curran, B., Sadjadi, J. — 2008-11-06

Ischemia-reperfusion (IR) injury is a major insult to postcapillary venules. We hypothesized that IR increases postcapillary venular hydraulic conductivity and that IR-mediated changes in hydraulic conductivity result from temporally and mechanistically separate processes. A microcannulation technique was used to determine hydraulic conductivity (L_p) in rat mesenteric postcapillary venules serially throughout ischemia (45 min) and reperfusion (5 h) induced by superior mesenteric artery occlusion and release. Mesenteric IR resulted in a biphasic increase in L_p. White blood cell (WBC) adhesion slowly increased with maximal adhesion corresponding to the second peak (P < 0.005). After IR, tissue was harvested for RT-PCR analysis of ICAM-1, E-selectin, and P-selectin mRNA. Intercellular adhesion molecule-1 (ICAM-1) mRNA in the gut showed the most significant upregulation. Quantitative real-time PCR revealed that ICAM-1 mRNA was upregulated 60-fold in the gut. An ICAM-1 antibody was therefore used to determine the effect of WBC adhesion on L_p during IR. ICAM-1 inhibition attenuated L_p during the first peak and completely blocked the second peak (P < 0.005). When rats were fed a tungsten diet to inhibit xanthine oxidase and then underwent IR, L_p was dramatically attenuated during the first peak and mildly decreased the second peak (P < 0.005). Inhibition of xanthine oxidase by oxypurinol decreased L_p during IR by over 60% (P < 0.002). Tempol, a superoxide dismutase mimetic, decreased L_p during IR by over 30% (P < 0.01). We conclude that IR induces a biphasic increase in postcapillary hydraulic conductivity. Reactive oxygen species impact both the first transient peak and the sustained second peak. However, the second peak is also dependent on WBC-endothelial cell adhesion. These serial measurements of postcapillary hydraulic conductivity may lead the way for optimal timing of pharmaceutical therapies in IR injury.

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

Chen, X., Pavlish, K., Benoit, J. N. — 2008-11-06

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or β-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC₂₀) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC₂₀ phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC₂₀ phosphorylation were determined. Reductions in MLC₂₀ phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC₂₀ phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC₂₀ phosphorylation in β-escin-permeabilized vessels. Collectively, our data demonstrate that MLC₂₀ phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.

Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

2008-11-06

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH₄)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH₄ and 7,8-dihydrobiopterin (BH₂) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH₄ synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH₄) increased BH₄ as well as BH₂, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH₄ after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

Nerve stimulation induced overflow of neuropeptide Y and modulation by angiotensin II in spontaneously hypertensive rats

Byku, M., Macarthur, H., Westfall, T. C. — 2008-11-06

The sympathetic nervous system and renin-angiotensin system are both thought to contribute to the development and maintenance of hypertension in experimental models such as the spontaneously hypertensive rat (SHR). We demonstrated that periarterial nerve stimulation (NS) increased the perfusion pressure (PP) and neuropeptide Y (NPY) overflow from perfused mesenteric arterial beds of SHRs at 4–6, 10–12, and 18–20 wk of age, which correspond to prehypertensive, developing hypertensive, and maintained hypertensive stages, respectively, in the SHR. NS also increased PP and NPY overflow from mesenteric beds of Wistar-Kyoto (WKY) normotensive rats. NS-induced increases in PP and NPY were greater in vessels obtained from SHRs of all three ages compared with WKY rats. ANG II produced a greater increase in PP in preparations taken from SHRs than WKY rats. ANG II also resulted in a greater increase in basal NPY overflow from 10- to 12-wk-old and 18- to 20-wk-old SHRs than age-matched WKY rats. ANG II enhanced the NS-induced overflow of NPY from SHR preparations more than WKY controls at all ages studied. The enhancement of NS-induced NPY overflow by ANG II was blocked by the AT₁ receptor antagonist EMD-66684 and the angiotensin type 2 receptor antagonist PD-123319. In contrast, ANG II greatly enhanced norepinephrine overflow in the presence of PD-123319. Both captopril and EMD-66684 decreased neurotransmitter overflow from SHR mesenteric beds; therefore, we conclude that an endogenous renin-angiotensin system is active in this preparation. It is concluded that the ANG II-induced enhancement of sympathetic nerve stimulation may contribute to the development and maintenance of hypertension in the SHR.

Aging and prostacyclin responses in aorta and platelets from WKY and SHR rats

2008-11-06

In spontaneously hypertensive rat (SHR) aorta, prostacyclin is an endothelium-derived contracting factor contributing to the endothelial dysfunction. This study was designed to determine whether the impairment of the prostacyclin response is influenced by aging and whether such a dysfunction is observed in platelets. Isometric tension was measured in aortic rings, and aggregation was studied in platelet-rich plasma taken from 3-, 6-, and 15-mo-old Wistar-Kyoto rats (WKY) and SHR. In aorta from 3- and 6-mo-old WKY, prostacyclin and beraprost [prostacyclin receptor (IP) agonists] produced relaxations that were enhanced by Triplion (thromboxane-prostanoid receptor antagonist). In 15-mo-old WKY, the relaxations to beraprost were maintained, but not those to prostacyclin. In SHR aorta, prostacyclin or beraprost produced no or minor relaxations, which, in younger SHR, were enhanced by Triplion. In both strains, the relaxations were inhibited by CAY-10441 (IP receptor antagonist). The relaxations to forskolin and isoproterenol were reduced with aging. When compared with those of WKY, the relaxations to isoproterenol were reduced in 3- but not in 6- or 15-mo-old SHR, whereas those to forskolin were consistently diminished at any given age. Whatever the age, prostacyclin and beraprost produced CAY-10441-sensitive inhibitions of ADP-induced platelet aggregation. Both agonists were more potent in SHR than in WKY. Therefore, in platelets from WKY and SHR, the IP receptor-dependent antiaggregant response is functional and maintained during aging. In aorta from WKY those responses are reduced by aging and, in SHR, are already compromised at 3 mo. This dysfunction of the IP receptor is only partially explained by a general dysfunction of the adenylate cyclase pathway.